Project Summary Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance levels are growing and methicillin-resistant S. aureus (MRSA) infections are more challenging to treat, resulting in increased burden on both patients and healthcare systems. Despite being such an effective pathogen, S. aureus can asymptomatically colonize approximately 20% of the healthy adult population, primarily in the nasal cavity and secondarily on the skin. There is a clear need to understand the transition from colonizer to invader, since the majority of S. aureus disease is the result of autoinfection from the colonized strain. Our recent findings indicate that the ArlRS TCS plays a critical role in the S. aureus transition to an invasive pathogen. The primary output of ArlRS is controlling the expression of MgrA, a cytoplasmic regulator that represses urease and large surface proteins and induces immune evasion factors. Our model is that S. aureus exhibits the traits of a commensal when MgrA levels are low, and exhibits the trait of an invasive pathogen when MgrA levels are high. To test this model, in Aim 1 we will determine the contribution of ArlRS to S. aureus adherence, colonization, and infection. Toward this end, we will compare WT, ?arlRS, and ?mgrA mutant strains using in vitro models of adherence and an in vivo model of skin colonization. Additionally, we will perform real-time in vivo imaging to track MgrA regulon expression, and carry out skin infection and immune evasion assessments of these strains. In Aim 2, we will perform a biochemical characterization of ArlS and identify of ArlR-dependent promoters. ArlR is a response regulator of the OmpR family, and the ArlR DNA- binding site and target promoters are unknown. The ArlS kinase has an N-terminal sensor domain with two membrane-spanning passes that flank a 14.1 kDa extracellular Cache domain. The goal of this aim is to determine the biochemical properties of ArlS and ArlR, which will provide critical insight into the mechanism of ArlS regulation and ArlR promoter targets. We will assess the kinetics of ArlS-ArlR phosphotransfer using mutants to determine the enzyme activity (kinase and phosphatase activity) that is important for regulating the ArlRS regulon. We will also use SELEX to identify the ArlR binding site and confirm target promoters with qRT- PCR and RNAseq. In Aim 3, we will perform molecular analysis of the ArlS sensing mechanism. We hypothesize that the ArlS extracellular Cache domain responds to an environmental cue that results in high urease and SasG expression to promote S. aureus colonization. To further investigate this mechanism, we will examine signal control over ArlS function using reporter strains, and we will make site-directed changes in ArlS Cache domain residues and assess function. We will also screen for additional ArlS Cache domain ligands from small-molecule libraries by thermal shift PCR assays and NMR. Discovering ligands that alter ArlRS function and prevent transition of S. aureus to an invasive pathogen could have potential in treating antibiotic-resistant infections.